Detection of mecA-containing Staphylococcus spp.

ABSTRACT

The invention provides methods to detect mecA-containing Staphylococcus spp. in biological samples using real-time PCR. Primers and probes for the detection of  S. aureus  are provided by the invention. Articles of manufacture containing such primers and probes for detecting mecA-containing Staphylococcus spp. are further provided by the invention.

TECHNICAL FIELD

[0001] This invention relates to bacterial diagnostics, and moreparticularly to detection of Staphylococcus spp. that contain mecAnucleic acid sequences.

BACKGROUND

[0002] Methicillin resistance in Staphylococcus aureus andcoagulase-negative staphylococcus (CoNS) is associated with the presenceof the mecA gene (Geha et al., 1994, J. Clin. Microbiol., 32:1768).Isolates of staphylococcus that carry the mecA gene produce a modifiedpenicillin-binding protein, PBP-2′, which confers high-level resistanceto all beta-lactams, including penicillins, semisyntheticpenicillinase-resistant congeners, penems, carbapenems, andcephalosporins. Isolates of staphylococcus that are mecA positive shouldbe considered resistant to all β-lactam antimicrobials.

SUMMARY

[0003] The invention provides for methods of identifying mecA-containingStaphylococcus spp. in a biological sample. Primers and probes fordetecting mecA-containing Staphylococcus spp. are provided by theinvention, as are kits containing such primers and probes. Methods ofthe invention can be used to rapidly detect the presence or absence ofmecA-containing Staphylococcus spp. from specimens for diagnosis ofStaphylococcus infection. Using specific primers and probes, the methodsof the invention include amplifying and monitoring the development ofspecific amplification products using fluorescence resonance energytransfer (FRET).

[0004] In one aspect of the invention, there is provided a method fordetecting the presence or absence of one or more mecA-containingStaphylococcus spp. in a biological sample from an individual. Themethod to detect mecA-containing Staphylococcus spp. includes performingat least one cycling step, which includes an amplifying step and ahybridizing step. The amplifying step includes contacting the samplewith a pair of mecA primers to produce a mecA amplification product if amecA nucleic acid molecule is present in the sample. The hybridizingstep includes contacting the sample with a pair of mecA probes.Generally, the members of the pair of mecA probes hybridize within nomore than five nucleotides of each other. A first mecA probe of the pairof mecA probes is typically labeled with a donor fluorescent moiety anda second mecA probe of the pair of mecA probes is labeled with acorresponding acceptor fluorescent moiety. The method further includesdetecting the presence or absence of FRET between the donor fluorescentmoiety of the first mecA probe and the acceptor fluorescent moiety ofthe second mecA probe. The presence of FRET is usually indicative of thepresence of one or more mecA-containing Staphylococcus spp. in thesample, while the absence of FRET is usually indicative of the absenceof a mecA-containing Staphylococcus spp. in the sample.

[0005] A pair of mecA primers generally includes a first mecA primer anda second mecA primer. A first mecA primer can include the sequence5′-AAA CTA CGG TAA CAT TGA TCG CAA C-3′ (SEQ ID NO: 1), and a secondmecA primer can include the sequence 5′-TCT TGT ACC CAA TTT TGA TCC ATTT-3′ (SEQ ID NO:2). A first mecA probe can include the sequence 5′-GTGGAA TTG GCC AAT ACA GGA ACA GCA TA-3′ (SEQ ID NO:3), and a second mecAprobe can include the sequence 5′-GAG ATA GGC ATC GTT CCA AAG AAT GTA-3′(SEQ ID NO:4).

[0006] In some aspects, one of the mecA primers can be labeled with afluorescent moiety (either a donor or acceptor, as appropriate) and cantake the place of one of the mecA probes.

[0007] The members of the pair of mecA probes can hybridize within nomore than two nucleotides of each other, or can hybridize within no morethan one nucleotide of each other. A representative donor fluorescentmoiety is fluorescein, and corresponding acceptor fluorescent moietiesinclude LC-Red 640, LC-Red 705, Cy5, and Cy5.5. Additional correspondingdonor and acceptor fluorescent moieties are known in the art.

[0008] In one aspect, the detecting step includes exciting the sample ata wavelength absorbed by the donor fluorescent moiety and visualizingand/or measuring the wavelength emitted by the acceptor fluorescentmoiety (i.e., visualizing and′/or measuring FRET). In another aspect,the detecting step includes quantitating the FRET. In yet anotheraspect, the detecting step can be performed after each cycling step(e.g., in real-time).

[0009] Generally, the presence of FRET within 45 cycles (e.g., 20, 25,30, 35, or 40 cycles) indicates the presence of a Staphylococcusinfection in the individual. In addition, determining the meltingtemperature between one or both of the mecA probe(s) and the mecAamplification product can confirm the presence or absence of amecA-containing Staphylococcus spp.

[0010] Representative biological sample include nasal swabs, throatswabs, feces, dermal swabs, lymphoid tissue, cerebrospinal fluid, blood(and blood in blood culture bottles), sputum, bronchio-alveolar lavage,bronchial aspirates, lung tissue, and urine. The above-described methodscan further include preventing amplification of a contaminant nucleicacid. Preventing amplification can include performing the amplifyingstep in the presence of uracil and treating the sample with uracil-DNAglycosylase prior to amplifying.

[0011] In addition, the cycling step can be performed on a controlsample. A control sample can include the same portion of the mecAnucleic acid molecule. Alternatively, a control sample can include anucleic acid molecule other than a mecA nucleic acid molecule. Cyclingsteps can be performed on such a control sample using a pair of controlprimers and a pair of control probes. The control primers and probes areother than mecA primers and probes. One or more amplifying stepsproduces a control amplification product. Each of the control probeshybridizes to the control amplification product.

[0012] In another aspect of the invention, there are provided articlesof manufacture, or kits. Kits of the invention can include a pair ofmecA primers, and a pair of mecA probes, and a donor and correspondingacceptor fluorescent moieties. For example, the first mecA primerprovided in a kit of the invention can have the sequence 5′-AAA CTA CGGTAA CAT TGA TCG CAA C-3′ (SEQ ID NO:1) and the second mecA primer canhave the sequence 5′-TCT TGT ACC CAA TTT TGA TCC ATT T-3′ (SEQ ID NO:2).The first mecA probe provided in a kit of the invention can have thesequence 5′-GTG GAA TTG GCC AAT ACA GGA ACA GCA TA-3′ (SEQ ID NO:3) andthe second mecA probe can have the sequence 5′-GAG ATA GGC ATC GTT CCAAAG AAT GT-3′ (SEQ ID NO:4).

[0013] Articles of manufacture can include fluorophoric moieties forlabeling the probes or the probes can be already labeled with donor andcorresponding acceptor fluorescent moieties. The article of manufacturecan also include a package insert having instructions thereon for usingthe primers, probes, and fluorophoric moieties to detect the presence orabsence of mecA-containing Staphylococcus spp. in a sample.

[0014] In yet another aspect of the invention, there is provided amethod for detecting the presence or absence of mecA-containingStaphylococcus spp. in a biological sample from an individual. Such amethod includes performing at least one cycling step. A cycling step caninclude an amplifying step and a hybridizing step. Generally, anamplifying step includes contacting the sample with a pair of mecAprimers to produce a mecA amplification product if a mecA nucleic acidmolecule is present in the sample. Generally, a hybridizing stepincludes contacting the sample with a mecA probe. Such a mecA probe isusually labeled with a donor fluorescent moiety and a correspondingacceptor fluorescent moiety. The method further includes detecting thepresence or absence of fluorescence resonance energy transfer (FRET)between the donor fluorescent moiety and the acceptor fluorescent moietyof the mecA probe. The presence or absence of fluorescence is indicativeof the presence or absence of mecA-containing Staphylococcus spp. insaid sample.

[0015] In one aspect, amplification can employ a polymerase enzymehaving 5′ to 3′exonuclease activity. Thus, the first and secondfluorescent moieties would be within no more than 5 nucleotides of eachother along the length of the probe. In another aspect, the mecA probeincludes a nucleic acid sequence that permits secondary structureformation. Such secondary structure formation generally results inspatial proximity between the first and second fluorescent moiety.According to this method, the second fluorescent moiety on a probe canbe a quencher.

[0016] In another aspect of the invention, there is provided a methodfor detecting the presence or absence of mecA-containing Staphylococcusspp. in a biological sample from an individual. Such a method includesperforming at least one cycling step. A cycling step can include anamplifying step and a dye-binding step. An amplifying step generallyincludes contacting the sample with a pair of mecA primers to produce amecA amplification product if a mecA nucleic acid molecule is present inthe sample. A dye-binding step generally includes contacting the mecAamplification product with a double-stranded DNA binding dye. The methodfurther includes detecting the presence or absence of binding of thedouble-stranded DNA binding dye into the amplification product.According to the invention, the presence of binding is typicallyindicative of the presence of one or more mecA-containing Staphylococcusspp. in the sample, and the absence of binding is typically indicativeof the absence of a mecA-containing Staphylococcus spp. in the sample.Such a method can further include the steps of determining the meltingtemperature between the mecA amplification product and thedouble-stranded DNA binding dye. Generally, the melting temperatureconfirms the presence or absence of mecA-containing Staphylococcus spp.Representative double-stranded DNA binding dyes include SYBRGreenI®,SYBRGold®, and ethidium bromide.

[0017] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol.

[0018] The details of one or more embodiments of the invention are setforth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages of the invention will be apparent fromthe drawings and detailed description, and from the claims.

DETAILED DESCRIPTION

[0019] A real-time assay for detecting mecA-containing Staphylococcusspp. in a biological sample that is more sensitive and specific thanexisting assays is described herein. The invention provides primers andprobes for detecting mecA-containing Staphylococcus spp. and articles ofmanufacture containing such primers and probes. The increasedsensitivity of real-time PCR for detection of mecA-containingStaphylococcus spp. compared to other methods, as well as the improvedfeatures of real-time PCR including sample containment and real-timedetection of the amplified product, make feasible the implementation ofthis technology for routine diagnosis of Staphylococcus infections inthe clinical laboratory.

[0020] The assay described herein decreases the time it takes to detectmethicillin resistance in staphylococcus from 2 to 3 days down to a fewhours by utilizing sensitive and rapid PCR and specific FRET probedetection technology in a real-time PCR format. The assay provides thepatient and physician with more rapid antibiotic susceptibilityinformation necessary for appropriate treatment of infections. Methodsof the invention can be used for rapid detection of the mecA geneassociated resistance in isolates of S. aureus and CoNS. For isolates ofS. lugdunensis, S. cohnii, S. saprophyticus, S. warneri, S. xylosus, theassay described herein may represent the most accurate means fordetermination of mecA-associated antibiotic resistance.

[0021] mecA Nucleic Acids and Oligonucleotides

[0022] The invention provides methods to detect mecA-containingStaphylococcus spp. by amplifying, for example, a portion of the mecAnucleic acid. mecA nucleic acid sequences other than those exemplifiedherein also can be used to detect mecA-containing Staphylococcus spp. ina sample and are known to those of skill in the art. The nucleic acidsequence of mecA is available (see, for example, GenBank Accession No.AB033763). The mecA sequence from 5 methicillin resistant S. aureus(MRSA) isolates and 5 methicillin resistant CoNS (MRCONS) isolates areidentical to the mecA sequence from S. aureus. Homologs to mecA havingapproximately 80% homology are found in several Staphylococcus species(Ito et al., 2001, Antimicrobial Agents and Chemotherapy, 45:1323-36).Such homologous mecA sequences are not detecting using the primers andprobes disclosed herein. Primers and probes can be designed, however,that would detect such mecA homologs.

[0023] Specifically, primers and probes to amplify and detect mecAnucleic acid molecules are provided by the invention. Primers thatamplify a mecA nucleic acid molecule, e.g., S. aureus mecA, can bedesigned using, for example, a computer program such as OLIGO (MolecularBiology Insights, Inc., Cascade, CO). Important features when designingoligonucleotides to be used as amplification primers include, but arenot limited to, an appropriate size amplification product to facilitatedetection (e.g., by electrophoresis), similar melting temperatures forthe members of a pair of primers, and the length of each primer (i.e.,the primers need to be long enough to anneal with sequence-specificityand to initiate synthesis but not so long that fidelity is reducedduring oligonucleotide synthesis). Typically, oligonucleotide primersare 15 to 30 nucleotides in length. As used herein, “mecA primers”refers to oligonucleotide primers that anneal specifically to mecAnucleic acid sequences and initiate synthesis therefrom underappropriate conditions.

[0024] Designing oligonucleotides to be used as hybridization probes canbe performed in a manner similar to the design of primers, although themembers of a pair of probes preferably anneal to an amplificationproduct within no more than 5 nucleotides of each other on the samestrand such that FRET can occur (e.g., within no more than 1, 2, 3, or 4nucleotides of each other). This minimal degree of separation typicallybrings the respective fluorescent moieties into sufficient proximitysuch that FRET occurs. It is to be understood, however, that otherseparation distances (e.g., 6 or more nucleotides) are possible providedthe fluorescent moieties are appropriately positioned relative to eachother (for example, with a linker arm) such that FRET can occur. Inaddition, probes can be designed to hybridize to targets that contain apolymorphism or mutation, thereby allowing differential detection ofmecA-containing Staphylococcus spp. or isolate based on either absolutehybridization of different pairs of probes corresponding to theparticular mecA-containing Staphylococcus spp. or isolate to bedistinguished or differential melting temperatures between, for example,members of a pair of probes and each amplification product correspondingto a mecA-containing Staphylococcus spp. or isolate to be distinguished.As with oligonucleotide primers, oligonucleotide probes usually havesimilar melting temperatures, and the length of each probe must besufficient for sequence-specific hybridization to occur but not so longthat fidelity is reduced during synthesis. Oligonucleotide probes aregenerally 15 to 30 nucleotides in length. As used herein, “meca probes”refers to oligonucleotide probes that specifically anneal to mecaamplification products.

[0025] Constructs of the invention include vectors containing a mecAnucleic acid molecule, e.g., S. aureus mecA, or a fragment thereof.Constructs of the invention can be used, for example, as controltemplate nucleic acid molecules. Vectors suitable for use in the presentinvention are commercially available and/or produced by recombinant DNAtechnology methods routine in the art. mecA nucleic acid molecules canbe obtained, for example, by chemical synthesis, direct cloning from amecA-containing Staphylococcus spp., or by PCR amplification. A mecAnucleic acid molecule or fragment thereof can be operably linked to apromoter or other regulatory element such as an enhancer sequence, aresponse element, or an inducible element that modulates expression ofthe mecA nucleic acid molecule. As used herein, operably linking refersto connecting a promoter and/or other regulatory elements to a mecAnucleic acid molecule in such a way as to permit and/or regulateexpression of the mecA nucleic acid molecule. A promoter that does notnormally direct expression of mecA can be used to direct transcriptionof a mecA nucleic acid using, for example, a viral polymerase, abacterial polymerase, or a eukaryotic RNA polymerase II. Alternatively,the mecA native promoter can be used to direct transcription of a mecAnucleic acid, respectively, using, for example, an RNA polymerase enzyme(e.g., RNA polymerase II). In addition, operably linked can refer to anappropriate connection between a mecA promoter or regulatory element anda heterologous coding sequence (i.e., a non-mecA coding sequence, forexample, a reporter gene) in such a way as to permit expression of theheterologous coding sequence.

[0026] Constructs suitable for use in the methods of the inventiontypically include, in addition to mecA nucleic acid molecules, sequencesencoding a selectable marker (e.g., an antibiotic resistance gene) forselecting desired constructs and/or transformants, and an origin ofreplication. The choice of vector systems usually depends upon severalfactors, including, but not limited to, the choice of host cells,replication efficiency, selectability, inducibility, and the ease ofrecovery.

[0027] Constructs of the invention containing mecA nucleic acidmolecules can be propagated in a host cell. As used herein, the termhost cell is meant to include prokaryotes and eukaryotes. Prokaryotichosts may include E. coli, Salmonella typhimurium, Serratia marcescensand Bacillus subtilis. Eukaryotic hosts include yeasts such as S.cerevisiae, S. pombe, Pichia pastoris, mammalian cells such as COS cellsor Chinese hamster ovary (CHO) cells, insect cells, and plant cells suchas Arabidopsis thaliana and Nicotiana tabacum. A construct of theinvention can be introduced into a host cell using any of the techniquescommonly known to those of ordinary skill in the art. For example,calcium phosphate precipitation, electroporation, heat shock,lipofection, microinjection, and viral-mediated nucleic acid transferare common methods for introducing nucleic acids into host cells. Inaddition, naked DNA can be delivered directly to cells (see, e.g., U.S.Pat. Nos. 5,580,859 and 5,589,466).

[0028] Polymerase Chain Reaction (PCR)

[0029] U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188disclose conventional PCR techniques. PCR typically employs twooligonucleotide primers that bind to a selected nucleic acid template(e.g., DNA or RNA). Primers useful in the present invention includeoligonucleotide primers capable of acting as a point of initiation ofnucleic acid synthesis within mecA sequences. A primer can be purifiedfrom a restriction digest by conventional methods, or it can be producedsynthetically. The primer is preferably single-stranded for maximumefficiency in amplification, but the primer can be double-stranded.Double-stranded primers are first denatured, i.e., treated to separatethe strands. One method of denaturing double stranded nucleic acids isby heating.

[0030] The term “thermostable polymerase” refers to a polymerase enzymethat is heat stable, i.e., the enzyme catalyzes the formation of primerextension products complementary to a template and does not irreversiblydenature when subjected to the elevated temperatures for the timenecessary to effect denaturation of double-stranded template nucleicacids. Generally, the synthesis is initiated at the 3′ end of eachprimer and proceeds in the 5′ to 3′ direction along the template strand.Thermostable polymerases have been isolated from Thermus flavus, Truber, T thermophilus, T aquaticus, T lacteus, T rubens, Bacillusstearothermophilus, and Methanothermus fervidus. Nonetheless,polymerases that are not thermostable also can be employed in PCR assaysprovided the enzyme is replenished.

[0031] If the Staphylococcus template nucleic acid is double-stranded,it is necessary to separate the two strands before it can be used as atemplate in PCR. Strand separation can be accomplished by any suitabledenaturing method including physical, chemical or enzymatic means. Onemethod of separating the nucleic acid strands involves heating thenucleic acid until it is predominately denatured (e.g., greater than50%, 60%, 70%, 80%, 90% or 95% denatured). The heating conditionsnecessary for denaturing template nucleic acid will depend, e.g., on thebuffer salt concentration and the length and nucleotide composition ofthe nucleic acids being denatured, but typically range from about 90° C.to about 105° C. for a time depending on features of the reaction suchas temperature and the nucleic acid length. Denaturation is typicallyperformed for about 30 sec to 4 min.

[0032] If the double-stranded nucleic acid is denatured by heat, thereaction mixture is allowed to cool to a temperature that promotesannealing of each primer to its target sequence on the mecA nucleicacid. The temperature for annealing is usually from about 35° C. toabout 65° C. Annealing times can be from about 10 secs to about 1 min.The reaction mixture is then adjusted to a temperature at which theactivity of the polymerase is promoted or optimized, i.e., a temperaturesufficient for extension to occur from the annealed primer such thatproducts complementary to the template nucleic acid are generated. Thetemperature should be sufficient to synthesize an extension product fromeach primer that is annealed to a nucleic acid template, but should notbe so high as to denature an extension product from its complementarytemplate (e.g., the temperature for extension generally ranges fromabout 40° to 80° C.). Extension times can be from about 10 secs to about5 mins. PCR assays can employ nucleic acid such as DNA or RNA, includingmessenger RNA (mRNA). The template nucleic acid need not be purified; itmay be a minor fraction of a complex mixture, such as Staphylococcusnucleic acid contained in human cells. DNA or RNA may be extracted froma biological sample such as nasal swabs, throat swabs, feces, dermalswabs, lymphoid tissue, cerebrospinal fluid, blood (and blood in bloodculture bottles), sputum, bronchio-alveolar lavage, bronchial aspirates,lung tissue, and urine by routine techniques such as those described inDiagnostic Molecular Microbiology: Principles and Applications (Persinget al. (eds), 1993, American Society for Microbiology, Washington D.C.).Nucleic acids can be obtained from any number of sources, such asplasmids, or natural sources including bacteria, yeast, viruses,organelles, or higher organisms such as plants or animals.

[0033] The oligonucleotide primers are combined with PCR reagents underreaction conditions that induce primer extension. For example, extensionreactions generally include 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mMMgCl₂, 0.001% (w/v) gelatin, 0.5-1.0 μg denatured template DNA, 50pmoles of each oligonucleotide primer, 2.5 U of Taq polymerase, and 10%DMSO). The reactions usually contain 150 to 320 μM each of dATP, dCTP,dTTP, dGTP, or one or more analogs thereof.

[0034] The newly synthesized strands form a double-stranded moleculethat can be used in the succeeding steps of the reaction. The steps ofstrand separation, annealing, and elongation can be repeated as often asneeded to produce the desired quantity of amplification productscorresponding to the target mecA nucleic acid molecule. The limitingfactors in the reaction are the amounts of primers, thermostable enzyme,and nucleoside triphosphates present in the reaction. The cycling steps(i.e., denaturation, annealing, and extension) are preferably repeatedat least once. For use in detection, the number of cycling steps willdepend, e.g., on the nature of the sample. If the sample is a complexmixture of nucleic acids, more cycling steps will be required to amplifythe target sequence sufficient for detection. Generally, the cyclingsteps are repeated at least about 20 times, but may be repeated as manyas 40, 60, or even 100 times.

[0035] Fluorescence Resonance Energy Transfer (FRET)

[0036] FRET technology (see, for example, U.S. Pat. Nos. 4,996,143,5,565,322, 5,849,489, and 6,162,603) is based on a concept that when adonor fluorescent moiety and a corresponding acceptor fluorescent moietyare positioned within a certain distance of each other, energy transfertakes place between the two fluorescent moieties that can be visualizedor otherwise detected and/or quantitated. Two oligonucleotide probes,each containing a fluorescent moiety, can hybridize to an amplificationproduct at particular positions determined by the complementarity of theoligonucleotide probes to the target nucleic acid sequence. Uponhybridization of the oligonucleotide probes to the amplification productat the appropriate positions, a FRET signal is generated. Hybridizationtemperatures can range from about 35° C. to about 65° C. for about 10secs to about 1 min.

[0037] Fluorescent analysis can be carried out using, for example, aphoton counting epifluorescent microscope system (containing theappropriate dichroic mirror and filters for monitoring fluorescentemission at the particular range), a photon counting photomultipliersystem or a fluorometer. Excitation to initiate energy transfer can becarried out with an argon ion laser, a high intensity mercury (Hg) arclamp, a fiber optic light source, or other high intensity light sourceappropriately filtered for excitation in the desired range.

[0038] As used herein with respect to donor and corresponding acceptorfluorescent moieties “corresponding” refers to an acceptor fluorescentmoiety having an emission spectrum that overlaps the excitation spectrumof the donor fluorescent moiety. The wavelength maximum of the emissionspectrum of the acceptor fluorescent moiety should be at least 100 nmgreater than the wavelength maximum of the excitation spectrum of thedonor fluorescent moiety. Accordingly, efficient non-radiative energytransfer can be produced therebetween.

[0039] Fluorescent donor and corresponding acceptor moieties aregenerally chosen for (a) high efficiency Förster energy transfer; (b) alarge final Stokes shift (>100 nm); (c) shift of the emission as far aspossible into the red portion of the visible spectrum (>600 nm); and (d)shift of the emission to a higher wavelength than the Raman waterfluorescent emission produced by excitation at the donor excitationwavelength. For example, a donor fluorescent moiety can be chosen thathas its excitation maximum near a laser line (for example,Helium-Cadmium 442 nm or Argon 488 nm), a high extinction coefficient, ahigh quantum yield, and a good overlap of its fluorescent emission withthe excitation spectrum of the corresponding acceptor fluorescentmoiety. A corresponding acceptor fluorescent moiety can be chosen thathas a high extinction coefficient, a high quantum yield, a good overlapof its excitation with the emission of the donor fluorescent moiety, andemission in the red part of the visible spectrum (>600 nm).Representative donor fluorescent moieties that can be used with variousacceptor fluorescent moieties in FRET technology include fluorescein,Lucifer Yellow, B-phycoerythrin, 9-acridineisothiocyanate, LuciferYellow VS, 4-acetamido-4′-isothio-cyanatostilbene-2,2′-disulfonic acid,7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin, succinimdyl1-pyrenebutyrate, and4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid derivatives.Representative acceptor fluorescent moieties, depending upon the donorfluorescent moiety used, include LC™-Red 640, LC™-Red 705, Cy5, Cy5.5,Lissamine rhodamine B sulfonyl chloride, tetramethyl rhodamineisothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate,fluorescein, diethylenetriamine pentaacetate or other chelates ofLanthanide ions (e.g., Europium, or Terbium). Donor and acceptorfluorescent moieties can be obtained, for example, from Molecular Probes(Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).

[0040] The donor and acceptor fluorescent moieties can be attached tothe appropriate probe oligonucleotide via a linker arm. The length ofeach linker arm is important, as the linker arms will affect thedistance between the donor and acceptor fluorescent moieties. The lengthof a linker arm for the purpose of the present invention is the distancein Angstroms (Å) from the nucleotide base to the fluorescent moiety. Ingeneral, a linker arm is from about 10 to about 25 Å. The linker arm maybe of the kind described in WO 84/03285. WO 84/03285 also disclosesmethods for attaching linker arms to a particular nucleotide base, andalso for attaching fluorescent moieties to a linker arm.

[0041] An acceptor fluorescent moiety such as an LC™-Red 640-NHS-estercan be combined with C6-Phosphoramidites (available from ABI (FosterCity, Calif.) or Glen Research (Sterling, Va.)) to produce, for example,LC™-Red 640-Phosphoramidite. Frequently used linkers to couple a donorfluorescent moiety such as fluorescein to an oligonucleotide includethiourea linkers (FITC-derived, for example, fluorescein-CPG's from GlenResearch or ChemGene (Ashland, Mass.)), amide-linkers(fluorescein-NHS-ester-derived, such as fluorescein-CPG from BioGenex(San Ramon, Calif.)), or 3′-amino-CPG's that require coupling of afluorescein-NHS-ester after oligonucleotide synthesis.

[0042] Detection of mecA-Containing Stayhylococcus spp.

[0043] Current phenotypic, culture-based methods require at least 48hours and frequently longer to detect β-lactam resistance associatedwith the mecA gene. Furthermore, the phenotypic evaluation ofmecA-associated resistance may lack sensitivity and specificity for somestaphylococcal species, e.g., S. lugdunensis. The “gold standard” testis a traditional agar or broth dilution test with dilutions ofantibiotics. These tests require 18 to 24 hours to perform. This inaddition to the time required for isolation of the organism fromclinical specimens, which may require an additional 24-48 hours.

[0044] A recent test, The Velogeneυ Genomic Identification Assay forMRSA, uses cycling probe technology (CPT) to give a visualidentification result in 90 minutes starting from a primary isolate.Velogene kits utilize a fluorescein labeled biotinylated DNA-RNA-DNAchimeric probe that binds to the complementary sequence of the mecA genefor MRSA. When the probe has hybridized with the target DNA, RNase Hcleaves the RNA portion of the chimeric probe, which results in afluorescein labeled fragment and a biotinylated fragment. The cleavedprobe disassociates from the target DNA allowing the probe cleavagecycle to be repeated. At the end of a 25-30 minute CPT reaction, theintact probe is detected in an ELISA format using a streptavidin-coatedmicrotiter plate with an anti-fluorescein horseradish peroxidaseconjugate. If complementary mecA target sequences are present in theisolate, all the probe molecules are cleaved and the ELISA well remainsclear. If no target is present, the intact probe is captured in theELISA well and the well turns blue from the action of horseradishperoxidase on TMB. The Velogene assay requires more hands on time thanthe rapid PCR test and does not have the potential for testing directlyfrom patient samples.

[0045] The invention provides an assay for detecting mecA-containingStaphylococcus spp. The LightCycler PCR assay is the first automated,real-time system for the detection of Staphylococcus mecA nucleic acid.The system is rapid (2-3 hours total sample preparation and analyticaltime), is sensitive (detects≧10 copies of mecA DNA/sample), specific(detects mecA DNA target exclusively), and has a wide dynamic linearrange of 10¹ to 10⁷ copies of mecA/sample. By using commerciallyavailable real-time PCR instrumentation (e.g., LightCyclerυ, RocheMolecular Biochemicals, Indianapolis, Ind.), PCR amplification anddetection of the amplification product can be combined in a singleclosed cuvette with dramatically reduced cycling time. Since detectionoccurs concurrently with amplification, the real-time PCR methodsobviate the need for manipulation of the amplification product, anddiminish the risk of cross-contamination between amplification products.Real-time PCR greatly reduces turn-around time and is an attractivealternative to conventional PCR techniques in the clinical laboratory.

[0046] The present invention provides methods for detecting the presenceor absence of one or more mecA-containing Staphylococcus spp. in abiological sample from an individual. Methods provided by the inventionavoid problems of sample contamination, false negatives, and falsepositives. The methods include performing at least one cycling step thatincludes amplifying a portion of a mecA nucleic acid molecule from asample using a pair of mecA primers, respectively. Each of the mecAprimers anneals to a target within or adjacent to a mecA nucleic acidmolecule such that at least a portion of each amplification productcontains nucleic acid sequence corresponding to mecA. More importantly,the amplification product should contain the nucleic acid sequences thatare complementary to the mecA probes. The mecA amplification product isproduced provided that mecA nucleic acid is present. Each cycling stepfurther includes contacting the sample with a pair of mecA probes.According to the invention, one member of each pair of the mecA probesis labeled with a donor fluorescent moiety and the other is labeled witha corresponding acceptor fluorescent moiety. The presence or absence ofFRET between the donor fluorescent moiety of the first mecA probe andthe corresponding acceptor fluorescent moiety of the second mecA probeis detected upon hybridization of the mecA probes to the mecAamplification product.

[0047] Each cycling step includes an amplification step and ahybridization step, and each cycling step is usually followed by a FRETdetecting step. Multiple cycling steps are performed, preferably in athermocycler. Methods of the invention can be performed using the mecAprimer and probe sets to detect the presence of mecA-containingStaphylococcus spp. As used herein, “mecA-containing Staphylococcusspp.” refers to Staphylococcus species that contain mecA nucleic acidsequences.

[0048] As used herein, “amplifying” refers to the process ofsynthesizing nucleic acid molecules that are complementary to one orboth strands of a template nucleic acid molecule (e.g., mecA nucleicacid molecules). Amplifying a nucleic acid molecule typically includesdenaturing the template nucleic acid, annealing primers to the templatenucleic acid at a temperature that is below the melting temperatures ofthe primers, and enzymatically elongating from the primers to generatean amplification product. Amplification typically requires the presenceof deoxyribonucleoside triphosphates, a DNA polymerase enzyme (e.g.,Platinum® Taq) and an appropriate buffer and/or co-factors for optimalactivity of the polymerase enzyme (e.g., MgCl₂ and/or KCl).

[0049] If amplification of mecA nucleic acid occurs and an amplificationproduct is produced, the step of hybridizing results in a detectablesignal based upon FRET between the members of the pair of probes. Asused herein, “hybridizing” refers to the annealing of probes to anamplification product. Hybridization conditions typically include atemperature that is below the melting temperature of the probes but thatavoids non-specific hybridization of the probes.

[0050] Generally, the presence of FRET indicates the presence of one ormore mecA-containing Staphylococcus spp. in the sample, and the absenceof FRET indicates the absence of mecA-containing Staphylococcus spp. inthe sample. Inadequate specimen collection, transportation delays,inappropriate transportation conditions, or use of certain collectionswabs (calcium alginate or aluminum shaft) are all conditions that canaffect the success and/or accuracy of a test result, however. Using themethods disclosed herein, detection of FRET within 45 cycling steps isindicative of a Staphylococcus infection.

[0051] Representative biological samples that can be used in practicingthe methods of the invention include nasal swabs, throat swabs, feces,dermal swabs, lymphoid tissue, cerebrospinal fluid, blood (and blood inblood culture bottles), sputum, bronchio-alveolar lavage, bronchialaspirates, lung tissue, and urine. Collection and storage methods ofbiological samples are known to those of skill in the art. Biologicalsamples can be processed (e.g., by nucleic acid extraction methodsand/or kits known in the art) to release Staphylococcus nucleic acid orin some cases, the biological sample can be contacted directly with thePCR reaction components and the appropriate oligonucleotides.

[0052] Melting curve analysis is an additional step that can be includedin a cycling profile. Melting curve analysis is based on the fact that anucleic acid sequence melts at a characteristic temperature called themelting temperature (Tm), which is defined as the temperature at whichhalf of the DNA duplexes have separated into single strands. The meltingtemperature of a DNA depends primarily upon its nucleotide composition.Thus, DNA molecules rich in G and C nucleotides have a higher Tm thanthose having an abundance of A and T nucleotides. By detecting thetemperature at which the FRET signal is lost, the melting temperature ofprobes can be determined. Similarly, by detecting the temperature atwhich signal is generated, the annealing temperature of probes can bedetermined. The melting temperature(s) of the mecA probes from therespective amplification product can confirm the presence or absence ofmecA-containing Staphylococcus spp. in the sample.

[0053] Within each thermocycler run, control samples are cycled as well.Positive control samples can amplify nucleic acid control template(e.g., a nucleic acid other than mecA) using, for example, controlprimers and control probes. Positive control samples can also amplify,for example, a plasmid construct containing a mecA nucleic acidmolecule. Such a plasmid control can be amplified internally (e.g.,within the sample) or in a separate sample run side-by-side with thepatients' samples. Each thermocycler run should also include a negativecontrol that, for example, lacks mecA template DNA. Such controls areindicators of the success or failure of the amplification, hybridizationand/or FRET reaction. Therefore, control reactions can readilydetermine, for example, the ability of primers to anneal withsequence-specificity and to initiate elongation, as well as the abilityof probes to hybridize with sequence-specificity and for FRET to occur.

[0054] In an embodiment, the methods of the invention include steps toavoid contamination. For example, an enzymatic method utilizinguracil-DNA glycosylase is described in U.S. Pat. Nos. 5,035,996,5,683,896 and 5,945,313 to reduce or eliminate contamination between onethermocycler run and the next. In addition, standard laboratorycontainment practices and procedures are desirable when performingmethods of the invention. Containment practices and procedures include,but are not limited to, separate work areas for different steps of amethod, containment hoods, barrier filter pipette tips and dedicated airdisplacement pipettes. Consistent containment practices and proceduresby personnel are necessary for accuracy in a diagnostic laboratoryhandling clinical samples.

[0055] Conventional PCR methods in conjunction with FRET technology canbe used to practice the methods of the invention. In one embodiment, aLightCycler™ instrument is used. A detailed description of theLightCycler™ System and real-time and on-line monitoring of PCR can befound at http://biochem.roche.com/lightcvcler. The following patentapplications describe real-time PCR as used in the LightCycler™technology: WO 97/46707, WO 97/46714 and WO 97/46712. The LightCycler™instrument is a rapid thermal cycler combined with a microvolumefluorometer utilizing high quality optics. This rapid thermocyclingtechnique uses thin glass cuvettes as reaction vessels. Heating andcooling of the reaction chamber are controlled by alternating heated andambient air. Due to the low mass of air and the high ratio of surfacearea to volume of the cuvettes, very rapid temperature exchange ratescan be achieved within the LightCycler™ thermal chamber. Addition ofselected fluorescent dyes to the reaction components allows the PCR tobe monitored in real time and on-line. Furthermore, the cuvettes serveas an optical element for signal collection (similar to glass fiberoptics), concentrating the signal at the tip of the cuvette. The effectis efficient illumination and fluorescent monitoring of microvolumesamples.

[0056] The LightCycler™ carousel that houses the cuvettes can be removedfrom the instrument. Therefore, samples can be loaded outside of theinstrument (in a PCR Clean Room, for example). In addition, this featureallows for the sample carousel to be easily cleaned and sterilized. Thefluorometer, as part of the LightCycler™ apparatus, houses the lightsource. The emitted light is filtered and focused by an epi-illuminationlens onto the top of the cuvette. Fluorescent light emitted from thesample is then focused by the same lens, passed through a dichroicmirror, filtered appropriately, and focused onto data-collectingphotohybrids. The optical unit currently available in the LightCycler™instrument (Roche Molecular Biochemicals, Catalog No. 2 011 468)includes three band-pass filters (530 nm, 640 nm, and 710 nm), providingthree-color detection and several fluorescence acquisition options. Datacollection options include once per cycling step monitoring, fullycontinuous single-sample acquisition for melting curve analysis,continuous sampling (in which sampling frequency is dependent on samplenumber) and/or stepwise measurement of all samples after definedtemperature interval.

[0057] The LightCycler™ can be operated using a PC workstation and canutilize a Windows NT operating system. Signals from the samples areobtained as the machine positions the capillaries sequentially over theoptical unit. The software can display the fluorescence signals inreal-time immediately after each measurement. Fluorescent acquisitiontime is 10-100 milliseconds (msec). After each cycling step, aquantitative display of fluorescence vs. cycle number can be continuallyupdated for all samples. The data generated can be stored for furtheranalysis.

[0058] As an alternative to FRET, an amplification product can bedetected using a double-stranded DNA binding dye such as a fluorescentDNA binding dye (e.g., SYBRGreenI® or SYBRGold® (Molecular Probes)).Upon interaction with the double-stranded nucleic acid, such fluorescentDNA binding dyes emit a fluorescence signal after excitation with lightat a suitable wavelength. A double-stranded DNA binding dye such as anucleic acid intercalating dye also can be used. When double-strandedDNA binding dyes are used, a melting curve analysis is usually performedfor confirmation of the presence of the amplification product.

[0059] As described herein, amplification products also can be detectedusing labeled hybridization probes that take advantage of FRETtechnology. A common format of FRET technology utilizes twohybridization probes. Each probe can be labeled with a differentfluorescent moiety and are generally designed to hybridize in closeproximity to each other in a target DNA molecule (e.g., an amplificationproduct). A donor fluorescent moiety, for example, fluorescein, isexcited at 470 nm by the light source of the LightCycler™ Instrument.During FRET, the fluorescein transfers its energy to an acceptorfluorescent moiety such as LightCycler™-Red 640 (LC™-Red 640) orLightCycler™-Red 705 (LC™-Red 705). The acceptor fluorescent moiety thenemits light of a longer wavelength, which is detected by the opticaldetection system of the LightCycler™ instrument. Efficient FRET can onlytake place when the fluorescent moieties are in direct local proximityand when the emission spectrum of the donor fluorescent moiety overlapswith the absorption spectrum of the acceptor fluorescent moiety. Theintensity of the emitted signal can be correlated with the number oforiginal target DNA molecules (e.g., the number of copies of mecA).

[0060] Another FRET format utilizes TaqMan® technology to detect thepresence or absence of an amplification product, and hence, the presenceor absence of mecA-containing Staphylococcus spp. TaqMan® technologyutilizes one single-stranded hybridization probe labeled with twofluorescent moieties. When a first fluorescent moiety is excited withlight of a suitable wavelength, the absorbed energy is transferred to asecond fluorescent moiety according to the principles of FRET. Thesecond fluorescent moiety is generally a quencher molecule. During theannealing step of the PCR reaction, the labeled hybridization probebinds to the target DNA (i.e., the amplification product) and isdegraded by the 5′ to 3′ exonuclease activity of the Taq Polymeraseduring the subsequent elongation phase. As a result, the excitedfluorescent moiety and the quencher moiety become spatially separatedfrom one another. As a consequence, upon excitation of the firstfluorescent moiety in the absence of the quencher, the fluorescenceemission from the first fluorescent moiety can be detected. By way ofexample, an ABI PRISM® 7700 Sequence Detection System (AppliedBiosystems, Foster City, Calif.) uses TaqMan® technology, and issuitable for performing the methods described herein for detectingmecA-containing Staphylococcus spp. Information on PCR amplification anddetection using an ABI PRISM® 770 system can be found athttp://www.appliedbiosystems.com/products.

[0061] Molecular beacons in conjunction with FRET also can be used todetect the presence of an amplification product using the real-time PCRmethods of the invention. Molecular beacon technology uses ahybridization probe labeled with a first fluorescent moiety and a secondfluorescent moiety. The second fluorescent moiety is generally aquencher, and the fluorescent labels are typically located at each endof the probe. Molecular beacon technology uses a probe oligonucleotidehaving sequences that permit secondary structure formation (e.g., ahairpin). As a result of secondary structure formation within the probe,both fluorescent moieties are in spatial proximity when the probe is insolution. After hybridization to the target nucleic acids (i.e.,amplification products), the secondary structure of the probe isdisrupted and the fluorescent moieties become separated from one anothersuch that after excitation with light of a suitable wavelength, theemission of the first fluorescent moiety can be detected.

[0062] It is understood that the present invention is not limited by theconfiguration of one or more commercially available instruments.

[0063] Articles of Manufacture

[0064] The invention further provides for articles of manufacture todetect mecA-containing Staphylococcus spp. An article of manufactureaccording to the present invention can include primers and probes usedto detect mecA-containing Staphylococcus spp., together with suitablepackaging materials. Representative primers and probes for detection ofmecA-containing Staphylococcus spp. are capable of hybridizing to mecAnucleic acid molecules. Methods of designing primers and probes aredisclosed herein, and representative examples of primers and probes thatamplify and hybridize to mecA nucleic acid molecules are provided.

[0065] Articles of manufacture of the invention also can include one ormore fluorescent moieties for labeling the probes or, alternatively, theprobes supplied with the kit can be labeled. For example, an article ofmanufacture may include a donor fluorescent moiety for labeling one ofthe mecA probes and an acceptor fluorescent moiety for labeling theother mecA probe. Examples of suitable FRET donor fluorescent moietiesand corresponding acceptor fluorescent moieties are provided above.

[0066] Articles of manufacture of the invention also can contain apackage insert or package label having instructions thereon for usingthe mecA primers and probes to detect Staphylococcus spp. in a sample.Articles of manufacture may additionally include reagents for carryingout the methods disclosed herein (e.g., buffers, polymerase enzymes,co-factors, or agents to prevent contamination). Such reagents may bespecific for one of the commercially available instruments describedherein.

[0067] The invention will be further described in the followingexamples, which do not limit the scope of the invention described in theclaims.

EXAMPLES Example 1 Oligonucleotide Primers and Probes

[0068] Primers and probes were designed using the OLIGO software(Molecular Biology Insights, Inc., Cascade, Oreg.). Primers weresynthesized on a 0.2 μM scale by the Mayo Molecular Biology CoreFacility (Rochester, Minn.). Probes were synthesized by TIB Molbiol LLC(Adelphia, N.J.), and were dissolved in TE′ (10 mM Tris (pH 8.0), 0.1 mMEDTA) to a final concentration of 20 μM. Sequences for primers andprobes are shown in Table 1. The GenBank Accession number for thereference sequence used to design the primers and probes for each targetis shown in Table 1.

Table 1. Primers and Probes for the Detection of S. aureus

[0069] TABLE 1 Primers and Probes for the Detection of S. aureus GeneBank Position Accession (Product Primer/ Gene Target # Size, bp) ProbeName Sequence mecA AB033763 315 mecA-F 5′-AAA CTA CGG TAA CAT TGA TCGCAA C-3′ (Forward Primer) (SEQ ID NO:1) mecA-R 5′-TCT TGT ACC CAA TTTTGA TCC ATT T-3′ (Reverse Primer) (SEQ ID NO:2) mecA-FL 5′-GTG GAA TTGGCC AAT ACA GGA ACA GCA TA-3′ (SEQ ID NO:3) mecA-RD 5′-GAG ATA GGC ATCGTT CCA AAG AAT GTA-3′ (SEQ ID NO:4)

[0070] Primers were adjusted to 50 μM by measuring the A₂₆₀ of a 1/50dilution (196 l water+4 μl, Dilution Factor (DF)=50). The concentrationwas estimated by the following formula:

(μM found/50)×μl remaining)−μl remaining=water to add

[0071] Probes were dissolved in TE′ to a concentration of 20 μM(supplied with the probes and resuspended according to manufacturer'sinstructions). The concentration of oligonucleotides and dye was doublechecked by UV absorption using the following equations from Biochemica,1999,1:5-8:$\lbrack{dye}\rbrack = {{\frac{A_{dye}}{E_{dye}}\quad\lbrack{oligo}\rbrack} = \frac{A_{260} - \left( {A_{260} \times \frac{E_{260{({dye})}}}{E_{dye}}} \right)}{\frac{10^{6}}{{nmol}\text{/}A_{260}}}}$

Absorbance Emission Abs max E_(dye) E_(260(dye)) Max Dye (nm) (M⁻¹cm⁻¹)(M⁻¹cm⁻¹) (nm) Fluorescein 494 68,000 2,000 524 LC Red 640 622 110,00031,000 638

[0072] Plasmid controls were produced by cloning the mecA productamplified by the mecA primers into the pCR® 2.1 TOPO® TA cloning vector(Invitrogen Corp., Carlsbad, Calif.). The recombinant vectors weretransformed into chemically competent TOP10 E. coli cells. The correctrecombinant plasmid was confirmed and purified with a Wizard MiniPrep(Promega Corp., Madison, Wiss.) Cleaning kit. The stock concentrationsof the controls (in genomic equivalents) were determined. The plasmidcontaining the mecA insert was used to determine the analyticalsensitivity of the assay. Plasmid concentration or the copy number ofthe gene target insert was determined with the following formula:

DS DNA, A₂₆₀ to molecules/μ

[0073] Given:

[0074] 1. (A₂₆₀×Dilution Factor)/20=mg/ml =μg/μl DS DNA

[0075] 1 A₂₆₀ =50 μg/ml

[0076] 1 A₂₆₀ (50)=μg/ml

[0077] 1 A₂₆₀ (50)/1000=μg/μl

[0078] 2. (6.02×10²³ molecules/mole)/(10¹² pmole/mole)=6.02×10¹¹molecules/pmole

[0079] 3. Base pairs of DNA in molecule=N

[0080] Then:

(A ₂₆₀ ×DF)/20 μg/μl×10⁶ pg/μg×1 pmol/660 pg×1/N×6.02×10¹¹molecules/pmole=molecules/μl

[0081] Shortcut calculation:

((A ₂₆₀ ×DF)/20)×(9.12×10¹⁴ /N)=molecules/μl

Example 2 PCR Conditions

[0082] A mechanical method of shaking with small beads was used to lysestaphylococcus from colonies, blood or blood culture media. Theadvantages of mechanical lysis are speed, simplicity and low cost. Thesensitivity was found to be less than 10 organisms per assay, suggestingadequate lysis.

[0083] Staphylococcus extraction buffer (SEB) (1 mM EDTA, 0.1 mM EGTA,0.1 mM TRIS (pH 8.0)) was used when extracting nucleic acid fromcolonies to inhibit nucleases from the organism, especially S. aureusthermonuclease. S. aureus thermonuclease prefers calcium as a cofactor,and the EGTA in the buffer chelates calcium and inhibits the enzyme.EDTA was used to bind magnesium, which is the cofactor for mostnucleases. The concentration of both chelators was low enough to haveminimal effect on the PCR reaction without additional sample cleanup.

[0084] The extracted nucleic acid from a colony had a very highconcentration of target DNA, so the potential for contamination washigher. When working with these types of samples, careful handling tocontain the target nucleic acids was necessary.

[0085] For extraction of nucleic acids from culture, the followingprotocol was followed. 500 μl of SEB was added to a bead tube (FastPrepLysing Matrix B, QBiogene, Inc. Catalog # 6911-100). A portion of abacterial colony was placed in the bead tube. The cap was replaced andthe sample processed using the FastPrep instrument (Qbiogene, Inc) for30 seconds at speed setting 6.0, or the tube placed into a DisruptorGenie (Scientific Industries) for 60 seconds. The tube was centrifugedfor 30 seconds at 12,000 to 20,000×g to pellet any particulate material.The upper supernatant was analyzed for mecA using the LightCycler™assay.

[0086] For extraction of nucleic acids from blood, blood culture media,and other specimens, the following method was used. The extraction wasconfirmed using the BD Bactec™ PLUS+ Aerobic/F blood culture media. 500μl blood or blood culture media was placed in a bead tube. Swabs wereswirled in the bead tube with 500 μl SEB. The cap was replaced and thesample was processed as described above using the FastPrep or theDisruptor Genie. The tube was centrifuged for 30 seconds at 12,000 to20,000×g. 200 μl was extracted using MagNAPure (Roche, Catalog 3 038505). Other extraction methods did not perform well with the bloodculture media. The extracted nucleic acid was analyzed for mecA by theLightCycler™ assay.

[0087] For the LightCycler™ assay, a 5 μl aliquot of extracted nucleicacid was added to 15 μl of LightCycler™ Master Mix in each reactioncapillary. A no-target control received 15 μl of LightCycler™ Master Mixwith 5 μl water. LightCycler ™ Master Mix-mecA Ingredient Stock Final μlWater — — 110 MgCl₂ 50 mM   3 mM 12 FastStart Reagent^(a) 10 X   1 X 20Primer-F&R 25 μM 0.5 μM 4 Probe-FL 20 μM 0.2 μM 2 Probe-RD 20 μM 0.2 μM2 Total volume 150

[0088] The PCR reagents and specimen extract are centrifuged in thecapillary to facilitate mixing. All capillaries are then sealed andamplified using the following protocol.

[0089] Quantification Settings:

[0090] Channel Settings F2

[0091] Experimental Protocol:

[0092] Melt (1 cycle) Type: none

[0093] 95° C., 10 min, 20°/sec slope

[0094] PCR(40 cycles) Type: Quantification

[0095] 95° C., 10 sec hold, 20° C./sec slope

[0096] 55° C., 10 sec hold, 20° C./sec slope, single acquisition

[0097] 72° C., 12 sec hold, 20° C./sec slope

[0098] Melt (1 cycle) Type: Melting Curve

[0099] 95° C., 10 sec hold, 20° C./sec slope

[0100] 60° C., 0 sec hold, 2° C./sec slope

[0101] 45° C., 30 sec hold, 0.2° C./sec slope

[0102] 85° C., 0 sec hold, 0.2° C./sec slope, continuous acquisition

[0103] Cool (1 cycle) Type: None

[0104] 40°, 30 sec hold, 20° C./sec slope

[0105] Amplification of the mecA sequences using the mecA primersdisclosed herein results in a 315 bp amplification product.

Example 3 Results. Assay Validation, and Quality Control

[0106] For the following control experiments, amplification wasperformed as described above in Examples 1 and 2.

[0107] Control experiments were performed to determine if the primersand probes described herein for detecting vancomycin-resistantenterococci cross-reacted with DNA from similar organisms or fromorganisms commonly found in the specimens. For the crossreactivitypanels, the presence of microorganism DNA was initially confirmed byamplification of 16S rRNA and electrophoretic separation of theamplification product (Johnson, 1994, Methods for General and MolecularBacteriology, American Society for Microbiology, Washington D.C.).Respiratory Specificity Panel Sample Organism Source mecA R2Acinetobacter lwoffli QC Strain negative R3 Aeromonas hydrophiliaCAP-D-1-82 negative R4 Bordetella bronchioseptica patient isolatenegative R5 Bordetella holmesii patient isolate negative R6 Bordetellaparapertussis ATCC 15311 negative R7 Bordetella pertussis ATCC 9797negative R8 Campylobacter jejuni CDC-AB2- negative C15-82 R10Corynebacterium patient isolate negative (Archanobacterium) haemolyticumR11 Corynebacterium dipheriae SCB-25-86 negative R12 CorynebacteriumNY-4-88 negative pseudodiptheriae R13 Eseherichia coli patient isolatenegative R14 Haemophilus influenza ATCC 49766 negative R15 Homo sapiensMRC-5 cells negative R16 Klebsiella oxytoca patient isolate negative R17Klebsiella pneumoniae patient isolate negative R18 Legionella jordanisATCC 33623 negative R19 Legionella pneumophila ATCC 33152 negative R20Listeria monocytogenes patient isolate negative R21 Moraxellacatarrhalis patient isolate negative R22 Morganella morganji CAP-D-5-79negative R23 Mycoplasma pneumoniae patient isolate negative R24Neiserria gonorrheae patient isolate negative R25 Neiserria meningitidispatient isolate negative R26 Proteus mirabilis patient isolate negativeP27 Proteus vulgaris patient isolate negative P28 Pseudomonas aeruginosaATCC 27853 negative P29 Pseudomonas cepacia patient isolate negative R30Pseudomonas fluorescens patient isolate negative R31 Staphylococcusaureus ATCC 25923 negative R32 Staphylococcus epidermidis patientisolate negative R33 Stenotrophomonas SOB-33-77 negative maltophilia R34Legionella micdadei ATCC 33204 negative R35 Citrobacter freundii patientisolate negative R36 Streptococcus pneumoniae ATCC 49619 negative R37Bordetella bronchioseptica ATCC 19395 negative R38 Streptococcuspyogenes patient isolate negative

[0108] Staphylococcus Specificity Panel Sample Organism Source mecA S1Staphylococcus aureus ATCC 25923 negative S3 Staphylococcus capitis ssp.capitis ATCC 35661 negative S4 Staphylococcus caprae patient isolatenegative S5 Staphylococcus cohnii ssp. cohnii ATCC 13509 negative S6Staphylococcus epidermidis MK 214 negative S7 Staphylococcushaemolyticus patient isolate negative S8 Staphylococcus hominis ssp.patient isolate negative horminus S9 Staphylococcus lugdunensis patientisolate negative S10 Staphylococcus saprophticus CAP-D-11-88 negativeS11 Staphylococcus sciuri ssp. sciuri ATCC 29060 negative S12Staphylococcus simulans patient isolate negative S13 Staphylococcuswarneri patient isolate negative S14 Staphylococcus xylosus ATCC 700404negative S15 Staphylococcus lentus ATCC 700403 negative S16 Rothia(Stomatococcus) mucilaginosa patient isolate negative

[0109] The respiratory and staphylococcus specificity panels did notshow cross-reactivity with the LightCycler™ mecA assay.

[0110] In addition, control experiments were performed to determine ifLightCycler™ amplification from clinical samples produced a singleamplification product. Amplification products were analyzed by 2%agarose gel electrophoresis. In positive clinical specimens,amplification using the LightCycler™ protocol generated a single band atthe expected size.

[0111] Additional control experiments were performed using dilutions ofpositive control plasmid to determine the sensitivity of theLightCycler™ assay. Plasmid dilutions were as follows. Stock to diluteμl stock μl diluent Copy/μl Copy/5 μl 2 × 10⁵/μl 100 900 2 × 10⁴/μl100,000 2 × 10⁴/μl 100 900 2 × 10³/μl 10,000 2 × 10³/μl 100 900 2 ×10²/μl 1000 2 × 10²/μl 100 900 2 × 10¹/μl 100 2 × 10¹/μl 100 900 1/μl 10

[0112] Data was plotted as the level of fluorescence detected relativeto the cycle number for each dilution value. The slope of the standardcurve was −3.269 with an r value=−0.99. Using the formulas ExponentialAmplification=10^((−1/slope)), and Efficiency=(10^((−1/slope)))−1, theefficiency of the reaction was determined to be 1.02. The sensitivity ofthe LightCycler™reaction was less than 50 copies of target per 5 μl ofsample. The efficiency of the reaction was >95%.

[0113] Further control experiments were performed to determine thesensitivity and specificity of the LightCycler™assay compared toculture-based methods. 50 patient isolates of MRSA and 50 methicillinsensitive S. aureus (MSSA) were randomly selected from patient'sisolates of staphylococcus archived at the Mayo Clinic (Rochester,Minn.) and sensitivities were determined using standard agar dilutionwith oxacillin. The same isolates were grown on sheep blood agar platesat 37° C. overnight, lysed with 0.5 ml of SEB with 250 μl of 0.1 μMzirconium beads in a 2 ml screw cap polypropylene tube, and processed ona Fast Prep instrument at a speed setting of 6 for 30 secs. The tube wascentrifuged for 1 min at 20,800 xg, and 5 μl of the supernatant wasanalyzed by the LightCycler™ assay described herein.

[0114] All MRSA samples were positive and all MSSA samples were negativeby the LightCycler™ mecA assay, thereby indicating that the LightCycler™assay provides 100% sensitivity and specificity. Initially, two isolatesof MRSA (928 & 987) were negative by the LightCycler mecA assay. Theagar dilution susceptibilities were repeated and the isolates were bothsensitive. It is presumed the mecA insertion sequence was lost uponfreezing and subculture, which is not uncommon.

[0115] Control experiments also were performed to determine theprecision (e.g., within-run, within-day, and between-day precision) ofthe LightCycler™ assay. Within-run precision of the LightCycler™ assaywas evaluated by assaying 5 μl of a positive control dilution 10 timeswithin the same amplification experiment. Within-day precision of theLightCycler™ assay was evaluated by assaying 5 μl of a positive controldilution 10 times during a single day. Between-day precision of theLightCycler™ assay was evaluated by assaying 5 μl of positive controldilution 10 times over a three-day period.

[0116] The average number of cycles at which FRET was detected in thewithin-run assays was 27.38±0.114; the average number of cycles at whichFRET was detected in the within-day assays was 27.12±0.075; and theaverage number of cycles at which FRET was detected in the between-dayprecision was 27.16±0.072. The precision of the average crossing pointmeasurement and the standard deviation was excellent.

[0117] Control experiments were performed to determine if theLightCycler™ assay produces the same results using 2, 5 or 10 μl of thenucleic acid sample extracted from a patient's sample. Mixes wereprepared for different target volumes essentially as described above,and 2 positive samples were tested at each volume. Similar results wereobtained from patient specimens when 2 μl, 5 μl, or 10 μl of sample wasused in the assay.

OTHER EMBODIMENTS

[0118] It is to be understood that while the invention has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention, which is defined by the scope of the appended claims.Other aspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for detecting the presence or absence of mecA-containing Staphyloccus spp. in a biological sample from an individual, said method comprising: performing at least one cycling step, wherein a cycling step comprises an amplifying step and a hybridizing step, wherein said amplifying step comprises contacting said sample with a pair of mecA primers to produce a mecA amplification product if a mecA nucleic acid molecule is present in said sample, wherein said hybridizing step comprises contacting said sample with a pair of mecA probes, wherein the members of said pair of mecA probes hybridize within no more than five nucleotides of each other, wherein a first mecA probe of said pair of mecA probes is labeled with a donor fluorescent moiety and said second mecA probe of said pair of mecA probes is labeled with a corresponding acceptor fluorescent moiety; and detecting the presence or absence of fluorescence resonance energy transfer (FRET) between said donor fluorescent moiety of said first mecA probe and said acceptor fluorescent moiety of said second mecA probe, wherein the presence of FRET is indicative of the presence of one or more mecA-containing Staphylococcus spp. in said sample, and wherein the absence of FRET is indicative of the absence of a mecA-containing Staphylococcus spp. in said sample.
 2. The method of claim 1, wherein said pair of mecA primers comprises a first mecA primer and a second mecA primer, wherein said first mecA primer comprises the sequence 5′-AAA CTA CGG TAA CAT TGA TCG CAA C-3′ (SEQ ID NO:1), and wherein said second mecA primer comprises the sequence 5′-TCT TGT ACC CAA TTT TGA TCC ATT T-3′ (SEQ ID NO:2).
 3. The method of claim 1, wherein said first mecA probe comprises the sequence 5′-GTG GAA TTG GCC AAT ACA GGA ACA GCA TA-3′ (SEQ ID NO:3), and wherein said second mecA probe comprises the sequence 5′-GAG ATA GGC ATC GTT CCAAAG AAT GTA-3′ (SEQ ID NO:4).
 4. The method of claim 1, wherein the members of said pair of mecA probes hybridize within no more than two nucleotides of each other.
 5. The method of claim 1, wherein the members of said pair of mecA probes hybridize within no more than one nucleotide of each other.
 6. The method of claim 1, wherein said donor fluorescent moiety is fluorescein.
 7. The method of claim 1, wherein said corresponding acceptor fluorescent moiety is selected from the group consisting of LC-Red 640, LC-Red 705, Cy5, and Cy5.5.
 8. The method of claim 1, wherein said detecting step comprises exciting said sample at a wavelength absorbed by said donor fluorescent moiety and visualizing and/or measuring the wavelength emitted by said acceptor fluorescent moiety.
 9. The method of claim 1, wherein said detecting comprises quantitating said FRET.
 10. The method of claim 1, wherein said detecting step is performed after each cycling step.
 11. The method of claim 1, wherein said detecting step is performed in real time.
 12. The method of claim 1, further comprising determining the melting temperature between one or both of said mecA probe(s) and said mecA amplification product, wherein said melting temperature confirms said presence or said absence of said mecA-containing Staphylococcus spp.
 13. The method of claim 1, wherein the presence of said FRET within 45 cycling steps is indicative of the presence of a Staphylococcus infection in said individual.
 14. The method of claim 1, wherein the presence of said FRET within 40 cycling steps is indicative of the presence of a Staphylococcus infection in said individual.
 15. The method of claim 1, wherein the presence of said FRET within 30 cycling steps is indicative of the presence of a Staphylococcus infection in said individual.
 16. The method of claim 1, further comprising: preventing amplification of a contaminant nucleic acid.
 17. The method of claim 16, wherein said preventing comprises performing said amplifying step in the presence of uracil.
 18. The method of claim 17, wherein said preventing further comprises treating said sample with uracil-DNA glycosylase prior to a first amplifying step.
 19. The method of claim 1, wherein said biological sample is selected from the group consisting of nasal swabs, throat swabs, feces, dermal swabs, lymphoid tissue, cerebrospinal fluid, blood, sputum, bronchio-alveolar lavage, bronchial aspirates, lung tissue, and urine.
 20. The method of claim 1, wherein said cycling step is performed on a control sample.
 21. The method of claim 20, wherein said control sample comprises said portion of said mecA nucleic acid molecule.
 22. The method of claim 1, wherein said cycling step uses a pair of control primers and a pair of control probes, wherein said control primers and said control probes are other than said mecA primers and mecA probes, wherein said amplifying step produces a control amplification product, wherein said control probes hybridize to said control amplification product.
 23. An article of manufacture, comprising: a pair of mecA primers; a pair of mecA probes; and a donor fluorescent moiety and a corresponding acceptor fluorescent moiety.
 24. The article of manufacture of claim 23, wherein said pair of mecA primers comprise a first mecA primer and a second mecA primer, wherein said first mecA primer comprises the sequence 5′-AAA CTA CGG TAA CAT TGA TCG CAA C-3′ (SEQ ID NO:1), and wherein said second mecA primer comprises the sequence 5′-TCT TGT ACC CAA TTT TGA TCC ATT T-3′ (SEQ ID NO:2).
 25. The article of manufacture of claim 23, wherein said pair of mecA probes comprises a first mecA probe and a second mecA probe, wherein said first mecA probe comprises the sequence 5′-GTG GAA TTG GCC AAT ACA GGA ACA GCA TA-3′ (SEQ ID NO:3), and wherein said second mecA probe comprises the sequence 5′-GAG ATA GGC ATC OTT CCAAAG AAT GTA-3′ (SEQ ID NO:4).
 26. The article of manufacture of claim 25, wherein said first mecA probe is labeled with said donor fluorescent moiety and wherein said second mecA probe is labeled with said corresponding acceptor fluorescent moiety.
 27. The article of manufacture of claim 23, further comprising a package insert having instructions thereon for using said pair of mecA primers and said pair of mecA probes to detect the presence or absence of mecA-containing Staphylococcus spp. in a sample.
 28. A method for detecting the presence or absence of mecA-containing Staphylococcus spp. in a biological sample from an individual or in a non-biological sample, said method comprising: performing at least one cycling step, wherein a cycling step comprises an amplifying step and a hybridizing step, wherein said amplifying step comprises contacting said sample with a pair of mecA primers to produce a mecA amplification product if a mecA nucleic acid molecule is present in said sample, wherein said hybridizing step comprises contacting said sample with a mecA probe, wherein the mecA probe is labeled with a donor fluorescent moiety and a corresponding acceptor fluorescent moiety; and detecting the presence or absence of fluorescence resonance energy transfer (FRET) between said donor fluorescent moiety and said acceptor fluorescent moiety of said mecA probe, wherein the presence or absence of fluorescence is indicative of the presence or absence of mecA-containing Staphylococcus spp. in said sample.
 29. The method of claim 28, wherein said amplification employs a polymerase enzyme having 5′ to 3′ exonuclease activity.
 30. The method of claim 29, wherein said first and second fluorescent moieties are within no more than 5 nucleotides of each other on said probe.
 31. The method of claim 30, wherein said second fluorescent moiety is a quencher.
 32. The method of claim 28, wherein said mecA probe comprises a nucleic acid sequence that permits secondary structure formation, wherein said secondary structure formation results in spatial proximity between said first and second fluorescent moiety.
 33. The method of claim 32, wherein said second fluorescent moiety is a quencher.
 34. A method for detecting the presence or absence of mecA -containing Staphylococcus spp. in a biological sample from an individual or in a non-biological sample, said method comprising: performing at least one cycling step, wherein a cycling step comprises an amplifying step and a dye-binding step, wherein said amplifying step comprises contacting said sample with a pair of mecA primers to produce a mecA amplification product if a mecA nucleic acid molecule is present in said sample, wherein said dye-binding step comprises contacting said mecA amplification product with a double-stranded DNA binding dye; and detecting the presence or absence of binding of said double-stranded DNA binding dye into said amplification product, wherein the presence of binding is indicative of the presence of one or more mecA-containing Staphylococcus spp. in said sample, and wherein the absence of binding is indicative of the absence of a mecA-containing Staphylococcus spp. in said sample.
 35. The method of claim 34, wherein said double-stranded DNA binding dye is selected from the group consisting of SYBRGreenI®, SYBRGold®, and ethidium bromide.
 36. The method of claim 34, further comprising determining the melting temperature between said mecA amplification product and said double-stranded DNA binding dye, wherein said melting temperature confirms said presence or absence of said mecA-containing Staphylococcus spp. 